Nucleic acid binding proteins include double-stranded DNA binding (dsDNA) proteins, single-stranded DNA (ssDNA) binding proteins and RNA binding proteins, etc.
dsDNA binding proteins are a group of proteins or protein molecule complexes which bind to specific sequences of dsDNA. dsDNA binding proteins include repressors and operator proteins of prokaryotes and transcription factors (TF) of eukaryotes, etc. These dsDNA binding proteins can activate, inhibit, reduce or enhance expression of target genes via binding to specific sequences of dsDNA (operator/promoter). In prokaryotes, the functions of repressor and operator proteins are relatively simple. They regulate genes encoding enzymes related to cellular metabolism and antibiotics resistance genes to adjust cellular physiological activity to the outside environment. In eukaryotes, transcription factors involve in many activities. Cell cycle, apoptosis, and tumorgenesis, etc., are all related to specific transcription factors. In biological system, especially in eukaryotic gene expression regulation network, expression of protein encoding genes includes: transcription activation via transcription factors, transcription, modification after transcription (splicing and 5′ and 3′ capping of RNA), translation, modification after translation (phosphorylation, glycosylation, acetylation, etc.), and is regulated at pre-transcriptional, post-transcriptional and post-translational levels.
Transcription activation via transcription factors is the first and important step in gene expression regulation network. Most of the stress reactions of an biological system to outside environment involve activation or turning off certain genes via specific transcription factors. Research indicates that expression of most of the eukaryotic genes is regulated by one or more specific transcription factors. More complicated organisms have more transcription factors and more complicated gene expression regulation mechanisms. As estimated, more than 5% protein encoding genes encode transcription factors. Many transcription factors are tightly related to cancers. For example, some transcription factors are only expressed in malignant tumors or can enhance expression of oncogenes (such as FOS and C-Myc); other transcription factors express weakly or do not express in malignant tumors (such as p53 and E2F). Thus, detecting levels of certain or all transcription factors in an organism at a certain time, combined with data of their target genes, allows obtaining regulation information before transcription. This information could be used for diagnosing tumorgenesis in a tissue, screening for drug target, studying mechanisms of cell stress responses, and observing activation and closing of cellular signal path, etc.
cDNA microarray technology is able to give mRNA profiling of all the transcription factor encoding genes of the genome. But only active transcription factors contribute to the regulation of gene expression. Activities of transcription factors usually are regulated by multiple protein modifications including phosphorylation, acetylation, glycosylation, etc. or intracellular localization of the transcription factors. Therefore, the quantities of active transcription factors do not always correlate with the quantities of transcription factors' in RNAs or proteins. For example, mRNA and protein expression level of the transcription factor Yin Yang 1 (YY1) are steady during cell cycle, but the level of the active YY1 changes greatly in different cell cycle stages. Thus, cDNA microarray technology cannot provide transcription factor expression information that researchers are interested in.
Conventional method for detecting “active” dsDNA binding protein is the gel shifting method (EMSA: Electrophoretic Mobility-Shift Assay, gel shift, band shift). Proteins to be tested are mixed with known dsDNA molecules labeled with radioisotopes. The reaction product is analyzed under polyacrylamide gel electrophoresis under non-denaturing condition. During electrophoresis, dsDNA molecules bound by proteins run slower than dsDNA molecules not bound by proteins. After electrophoresis, the result could be read by a autoradiography. On the film, separated electrophoresis bands could be seen and is used to detect binding between dsDNA and nucleic acid binding proteins. Recently, gel shifting technology has been improved. For example, fluorescence has been used to substitute radioisotopes. To solve nonspecific binding, antibody specific for dsDNA binding protein is used to detect DNA-protein complex. This method is called Super-shift. Gel shifting method has facilitated research in interaction between DNA and protein. However, it has obvious disadvantages: it involves complicated procedures; it is time and labor consuming (the experiment takes a whole day); it is low throughput (only one dsDNA binding protein is detected at a time); it requires large volume of sample if multiple dsDNA binding proteins are to be detected; it is hazardous to human if radioisotopes are used; and it is expensiveness if chemical or fluorescent dye is used.
Mercury™ transcription factor kit is a product from BD Biosciences Clontech Inc. (Palo Alto, Calif.). This kit provides a 96-well plate for detection of transcription factors. dsDNA probes which can be bound specifically by a transcription factor are immobilized onto the inner surface of each well. After a protein sample is added into the well, the immobilized dsDNA probes will bind to the corresponding transcription factors in the sample. After washing, primary antibody that specifically recognizes the transcription factor and enzyme-labeled secondary antibody which specifically recognizes the primary antibody are added one by one. Chemical dye is used for assay detection. This method is faster than the traditional EMSA method, and chemical dye is used instead of bio-hazardous radioisotopes. But this method is still a low-throughput method, and only one transcription factor could be examined at any one time in one well. Large volume of sample is needed for detecting multiple dsDNA binding proteins. There is a need for transcription factor specific antibodies and most of the transcription factor antibodies are not commercially available.
There are some sequence-specific ssDNA binding proteins and RNA binding proteins which regulate physiological activities in biological systems. More and more “antibody-like” aptamers which can specifically bind to target protein molecules are acquired by in vitro evolution method in recent years. There are no high-throughput methods for detecting these nucleic acid binding proteins.